verotoxins (VT1 and VT2c) to their specific cell surface receptor, globotriaosylceramide (Gb3) in the presence of auxiliary lipids both in a microtitre plate surface ...
Glycoconjugate Journal ( 1 9 9 6 ) 1 3 : 1 5 9 - 1 6 6
Influence of phospholipid chain length on verotoxin/ globotriaosyl ceramide binding in model membranes: comparison of a supported bilayer film and liposomes SARA
ARAB
1 and
CLIFFORD
A.
LINGWOOD
1'2.
Department of Microbiology, Research Institute, Hospital for Sick Children, 555 University Ave, Toronto, Ontario M5G 1 X8, Canada and Departments of 1Clinical Biochemistry, 2Biochemistry and Microbiology University of Toronto, Ontario, Canada Received 23 May 1995, revised 5 July 1995
The importance of the surrounding lipid environment on the availability of glycolipid carbohydrate for ligand binding was demonstrated by studying the influence of phosphatidylcholine fatty acid chain length on binding of verotoxins (VT1 and VT2c) to their specific cell surface receptor, globotriaosylceramide (Gb3) in the presence of auxiliary lipids both in a microtitre plate surface bilayer film and in a liposome membrane model system. In the microtitre assay, both VT1 and VT2c binding to Gb3 was increased as a function of decreasing PC acyl chain length likely resulting in increased Gb3 exposure. In the liposome assay VT1 binding was similarly modulated, however the effect on VT2c binding was more complex and did not follow a simple function of increased carbohydrate exposure. Earlier work established that C22:1 and C18:IGb3 fatty acid homologues were the preferred Gb3 receptor isoforms in the microtitre assay for VT1 and VT2c respectively. This selectivity was maintained in C16PC containing liposomes, but in C14PC liposomes, binding to C22:1Gb 3 (but not C18:1Gb3) was elevated such that this Gb3 species now became the preferred receptor for both toxins. This change in verotoxin/Gb 3 homologue binding selectivity in the presence of C14PC did not occur in the microtitre bilayer format. These results are consistent with our proposal that these toxins recognize different epitopes on the Gb3 oligosaccharide. We infer that relative availability of these epitopes for toxin binding in an artificial bilayer is influenced not only by the exposure due to the discrepancy between the fatty acyl chain lengths of Gb3 and PC, but by the physical mode of presentation of the bilayer s~ucmre. Such acyl chain length differences have a more marked effect in a supported bilayer film whereas only the largest discrepancies affect Gb3 receptor function in liposomes. The basis of phospholipid modulation of glycolipid carbohydrate accessibility for receptor function is likely complex and will involve phase separation, gel/liquid crystalline transition, packing and lateral mobility within the bilayer, suggesting that such parameters should be considered in the assessment of glycolipid receptor fimction in cells.
Keywords: glycolipid recognition, carbohydrate exposure, conformation, solid phase binding
Introduction Verotoxins (VT), also known as Shiga-like toxins [1], comprise a family o f toxins that are produced by certain strains of enterohaemorrhagic E. coli [2, 3]. Verotoxins are composed of an active (A) subunit and five binding (B) subunits [4, 5]. The family comprises four members, VT1 (identical to Shiga toxin), VT2, VT2c found in human E. coli isolates, and the toxin responsible for pig oedema disease, VT2e. The human toxins are the cause of two significant diseases, haemorrhagic colitis [6,7] and *To whom correspondence should be addressed. 0282-0080 © 1996 Chapman & Hall
haemolytic uraemic syndrome [8] which is the major cause o f acute pediatric renal failure. Both these diseases are characterized by microvascular endothelial cell damage and verotoxins are cytotoxic for certain endothelial cells in vitro [9, 10]. Globotriaosyl ceramide (galactoseo~ 1-4galactose/314glucosyl ceramide: Gb3) is the functional receptor for verotoxins (11-13). The glycolipid binding specificity is a primary determinant of the sites o f pathology in animal studies [14, 15]. VT binding is dependent on the terminal galactoseo~l-4galactose disaccharide [11, 12] but is also strongly influenced by the nature of the lipid moiety [16-18].
Arab and Lingwood
160 Internalization of cell-bound toxin occurs by receptormediated endocytosis [19], and is targeted intracellularty to the rough endoplasmic reticulum by a process termed retrogade transport [20-22]. The A subunit of VT1 is proteolytically nicked and reduced to the A1 fragment, the RNA glycanase activity of which suppresses protein synthesis through the inhibition of elongation factor 1 dependent aminoacyl-tRNA binding to the 60S ribosome [23]. However, cytotoxic potential of the holotoxin is also defined by B subunit Gb3 binding [5]. While the presence of Gb3 is necessary to confer susceptibility to VT cytotoxicity, the cellular concentration of Gb3 is not necessarily proportional to that sensitivity [24, 25]. Thus factors in addition to receptor concentration determine sensitivity to VT. It has been demonstrated that 'exposure' of glycosphingolipid carbohydrate is influenced by their lipid composition and membrane environment [26-31]. Such effects have been ascribed to the ability of the lipid matrix to modulate glycolipid head group exposure according to the relative phospholipid hydrocarbon chain length. The ability of glycolipids to phase separate into domains with a phospholipid bilayer can also affect carbohydrate availability to soluble ligands [32]. Previous studies have established that fatty acid heterogeneity [16], chain length and unsaturation [17] markedly affect verotoxin binding to Gb3 in a model phospholipid bilayer surface. Two members of the verotoxin family, VT1 and VT2c which show a significant difference in specific cytotoxicity [5], were found to bind preferentially to different Gb3 fatty acid isoforms under these conditions [17]. Conformational modelling studies of globoseries glycosphingolipids [33,34] suggest that the orientation and steric presentation of the saccharide chain at the membrane surface for recognition by antibodies or bacterial adhesins is determined, in part, by the combined influence of the linkage between the saccharide head group and the ceramide moiety and the relative plane of the membrane/solvent interface. In a study of synthetic glycolipids, we found that species which were not recognized by VT in tlc overlay could serve as effective receptors when presented in a lipid matrix [18]. Thus the lipid moiety of a glycolipid and the membrane environment can have a major effect on glycolipid carbohydrate receptor function. The interaction of the E. coli elaborated verotoxin with Gb3 provides a highly manipulable model to study the molecular basis of specific GSLs recognition. In order to approach the question whether our present solid phase assays accurately reflect the regulation of glycolipid receptor function in cells, we have compared verotoxin/ Gb3 binding in phospholipid/cholesterol bilayers in liposomes and as a surface film in microtitre wells. The influence of phospholipid hydrocarbon chain length
on the presentation of Gb3 in these bilayer formats for VT1 and VT2c binding was compared.
Materials and methods
Lipids Egg phosphatidylcholine (egg PC), dimyristoylphosphatidylcholine (diC14 PC), dipalmitoylphosphatidylcholine (diC16 PC), distearoylphosphatidyleholine (diC18 PC), dibehecicphosphatidylcholine (diC22 PC), fatty acids and cholesterol were purchased from Sigma. Extraction of human renal Gb3 Gb3 was purified as previously using human kidney obtained at autopsy [16]. Semisynthetic Gb3 homologues Semi synthetic Gb3 homologues containing C22:1 or C18:1 fatty acids were synthesized from lyso-Gb3 [35] as previously described [17]. Toxin preparation VT1 was purified from the over-producing recombinant E. coli strain pJLB28 [36] by the method involving polymxin B extraction, ultrafiltration, hydroxyapatite chromatography, chromatofocusing, and Cibacron Blue chromatography [5]. VT2c was purified by virtually the same method from an E. eoli clinical isolate, strain E 32511, which was determined to produce VT2c only [37]. VT1 and VT2e iodination Purified toxins (50-100 gg) were iodinated using Iodobeads (Pierce). Two iodobeads were incubated with 0.5 mCi Na 12sI (Amersham) and 100 gt of 0.1 M sodium phosphate, pH 7.2, for 10 rain. Labelled VTs were separated from unreacted iodine using a G-25 column equilibrated with PBS. VT glycolipid TLC overlay VT binding was performed as described by Yiu and Lingwood [38]. Microtitre verotoxin binding assay Based on the method used previously [16, 17] 100 gl of Gb3 (0.1 gg) in a methanolic solution containing egg PC (or C14 PC, C16 PC, C18 PC, or C22 PC) and cholesterol (1:5:2.5 by weight) was added in triplicate fo 96 well flexible microtitre plates and the solution was allowed to evaporate overnight. The plates were blocked with 2% BSA in 50 mM TBS (pH 7.4) for 1 h at room temperature. The wells were washed once with 0.1% BSA in TBS.
Verotoxin/globotriaosyl ceramide binding in model membranes Dilutions containing 50-300 ng of a25I-VT1 or a25I-VT2c diluted in 0.1% BSA in TBS were added to the Gb3coated wells and incubated for 2 h at room temperature. After five washes with 0.1% BSA in TBS, the wells were cut out and counted in a Beckman 5500 gamma-counter. Control wells containing no Gb3 were prepared and counted as above. Binding for controls was < 5% of total binding in the presence of Gb3 and was subtracted. Assays were performed in triplicate.
161
Results B I N D I N G OF H U M A N R E N A L Gb3 BY VT1 AND V T 2 c IN D I F F E R E N T PC C O N T A I N I N G LIPID BILAYERS Two binding assay systems were used to determine the effect of different fatty acid chain lengths of PC (C14, 16, 18, 22, or egg PC) on VT1 and VT2c recognition of Gb3 in a phospholipid bilayer.
a) Microtitre binding assay Preparation of Gb3 containing liposomes Multilamellar vesicles containing PC/Chol/GSLs (egg PC, C14 PC, C16 PC, C18 PC, or C22 PC) were used in order to avoid the high surface curvature of small unilamellar liposomes and the possible exclusion of the Gb3 during preparation of large unilamellar liposomes by extrusion or solvent injection procedures [39]. A chloroform:methanol (2:i v/v) solution of the lipid mixtures in weight ratio of 1:5:2.5 of Gb3:PC:Chol (as used in the microtitre plate binding assay) were added to a screwcapped glass tube, and after evaporation of the solvent under stream of nitrogen, was further dried under vacuum for 1 h. Then the lipids were dispersed in 50 mM Tris buffered saline (TBS) pH 7.4 at a concentration of 10 ng gl- 1 Gb3 by vortexing. The suspension was dispersed by alternate vigourous vortexing and heating for 30 s at a temperature of 9095 °C (5-10 °C above the lipid transition temperature) for a period of 10min. Background binding to control liposomes lacking Gb3, routinely > 6%, was subtracted. Measurements were made in triplicate.
Electron microscopy Gb 3 containing phospholipid liposomes were prepared as above, fixed with glutaraldehyde and osmium tetroxide, dehydrated, embedded and ultrathin sections visualized by negative staining with uranyl acetate [40].
VT-binding assay-Gb3 liposomes One hundred gl of freshly prepared liposomes containing 1 gg Gb3 were added in triplicate to 2 ml centrifuge tubes. The liposomes were blocked by incubation with 2% BSA in 50 mM TBS for 1 h at room temperature and washed once with TBS containing 0.2% BSA by centrifugation at 16000 × g for 30rains. Different concentrations of 125IVT1 or VT2c were added to each series in order to reach saturation and were incubated for 1 h at room temperature. The vesicles were washed four times with 0.2% BSA in TBS. Bound VT in the liposome pellet was counted using a gamma counter. All toxin binding experiments were repeated three times. One representative experiment with the means of triplicate determinations is shown in each case.
The binding of both VT1 and VT2c to a fixed concentration of Gb3 immobilized in a PC bilayer film on the microtitre plate surface was reduced as a function of increasing PC chain length (the binding order was C14 > C16 > C18 = C22). Binding was increased for C14.PC 3 fold as compared with C22/C1~8 PC (Fig. IA,
B).
b) Liposome binding assay A representative example of the multilamellar liposomes as determined by electron microscopy is shown in Fig. 2. The surface area of the liposomes did not vary significantly between the different preparations. For each toxin, overall binding was increased two-tothree-fold as compared to the microtitre assay. Increasing PC chain length had a different effect on the binding of either toxin to liposomes containing Gb3 (Fig. 1C, D) compared with the microtitre plate format. For VT1 (and VT2 not shown) binding was increased for C14PC containing liposomes, but increasing the PC chain length beyond C16 had no additional effect (Fig. 1C). The binding of renal Gb3 by VT2c in this model membrane system showed a distinct modulatory effect as compared to VT1 (and VT2, not shown) and did not follow a predictable trend. C18PC containing vesicles showed the maximum binding to VT2c (Fig. 1D), slightly higher than C14PC liposomes. Liposomes containing C22, C16 or egg PC showed lower binding (C18PC>C14>>C22>C16=egg PC) and were saturated at a lower VT2c concentration as compared to C18 and C14 vesicles. B I N D I N G OF S E M I S Y N T H E T I C Gb3 BY VT1 A N D V T 2 c IN D I F F E R E N T PC C O N T A I N I N G LIPID BILAYERS
a) Microtitre binding assay Semisynthetic Gb 3 homologues containing C22:1 or C 18:1 fatty acid (which are the preferred receptors in the bilayer film microtitre assay for VT1 and VT2c respectively [17]) were prepared from lyso-Gb3 as described in the Methods. The preferential binding of VT1 and VT2c was confirmed in the microtitre format. Binding of both toxins to the Gb 3 isoforms was increased in the presence of C14 PC relative
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Figure 1. Comparison of the effect of PC chain length on verotoxin binding to Gb:~ in the microtitre bilayer film (A, B) and liposomes (C, D). PC species: (.--O--): C14, ( - - [ ~ - - ) : 16, ( - - 0 - - ) : 18, ( - - A - - ) : 22, (." II.-.): egg PC, A, C: VT1, B, D: VT2c. Standard errors of triplicate determinations are shown.
to C16 PC but in each case maximum binding of VT1 and VT2c was to Gb3 containing C22:1 and C18:1 fatty acids respectively (Fig. 3A, B).
b) Liposomes The binding selectivity of VT1 and VT2c for the Gb 3 C22:1 and C18:1 fatty acid homologues was then compared in the multilamellar liposomes. The highest VT1 binding was observed for the C22:1 containing Gb3 in both a C14 PC environment and in C16 PC liposomes, as observed in the microtitre format above. Liposomal binding for these homologues showed a different effect for VT2c. For VT2c, the C18:1 Gb3 homologue was the best receptor as compared to C22:1 Gb3 when in C16 PCcontaining liposomes but the C22:1 homologue was the preferred VT2c receptor in C14 PC-containing liposomes. In this case the lipid environment not only influenced the binding of VT2c to Gb 3, but the fatty acid homologue
specificity was also altered. Essentially for both VT1 and VT2c, only binding to the C22:1Gb3 homologue is sensitive to changes in PC chain length in liposomes (Fig. 3C, D). VT B I N D I N G S T O I C H I O M E T R Y TO N A T U R A L Gb3 IN L I P O S O M E S The maximum binding was observed by VT1 (Fig. 1C) with natural Gb 3 in vesicles containing C14 PC. The capacity of liposomes containing 1 umol of Gb 3 to be bound by VT is approximately 4 pmol (or 1000:4, Gb3:VT mol/mol). VT1 is pentavalent and if all the valencies are bound, this result would suggest that 1:50 Gb3 are bound by the toxin. In addition, if the maximum exposure of Gb3 in the outer monolayer of the multilamellar vesicles is about 10% [30], this means 1:5 Gb3 are bound by the toxin in liposomes containing C14 PC.
Verotoxin/globotriaosyl ceramide binding in model membranes
163
Figure 2. Electron microscopy of multilamellar liposomes. (A) renal Gb3 in C18 PC, (228000×). (B) renal Gb3 in Ct4 PC (236000×).
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Figure 3. Comparison of the effect of PC chain length on VT/Gb 3 fatty acid homologue binding preference in the microti,tre bilayer film (A, B) or liposomes (C, D). C18:1 Gb3 and C 1 4 P C ( - - O - - ) , C18:1 Gb3 and C 1 6 P C ( - - O ~ ) ; C22:1 Gb3 and C 1 4 P C ( . . . . r~ . . . . ), c22:1 Gb3 and C16 PC ( . . . . • . . . . ). A, C:VT1, B, D: VT2c. Standard errors of triplicate determinations are shown.
Discussion The use of phospholipid/cholesterol mixtures as a manipulable model of plasma membrane function is common. While the composition of the Gb3/phospholipid matrices used does not reflect that of biological membranes, the present studies follow on from our demonstration of the importance of the lipid environment on toxin binding [18] and represent a first step to extrapolate in vitro solid phase binding to the regulation of glycolipid function in the plasma membrane of susceptible cells. Verotoxin glycolipid binding specificity was established by tic overlay [11, 41, 42], but although such results in general correlate with cell cytotoxicity there are several exceptions [25,38,43] and toxin binding in the solid phase has been shown to vary widely according to the manner of glycolipid immobilization [17, 18]. Thus the interaction of verotoxin with cell surface Gb3 is likely a complex procedure belied by its ease of assay in vitro. These studies show that for VT1, binding to Gb 3 in the
presence of phosphatidyl choline and cholesterol is modulated by the PC hydrocarbon chain length. This result is in agreement with several previous studies [29, 30] which demonstrate that glycolipid exposure for ligand binding in such a lipid environment is promoted by reduction of the PC chain length. The differential effect of PC chain length on the binding of VT2c (as opposed to VT1) to Gb3 containing liposomes supports our previous hypothesis that these two toxins bind to differing carbohydrate epitopes on the Gb3 molecule [17,44]. Binding of VT2c is not sglely a function of carbohydrate exposure, increased as a reciprocal of PC chain length. These results are also consistent with our recent thermodynamic calculation of two potential independent Gb3 binding sites per B subunit monomer [45]. Such sites may be differentially utilized by VT1 and VT2c. The finding that VT2c binding to Gb3 is modulated by PC chain length in a manner identical to VT1 in the
165
Verotoxin/globotriaosyl ceramide binding in model membranes microtitre format, provides evidence that the presentation of glycotipid carbohydrate in these two bilayer formats are not equivalent. The fact that for both VT1 and VT2c, only binding to C22:1, as opposed to C18:1 Gb3, was increased by reduction in PC fatty acyl chain length (Fig. 3C, D) in liposomes, resulted in a change in Gb 3 homologue binding selectivity for VT2c in C14 PC liposomes. This indicates that the membrane environment can exert a differential effect on glycolipid carbohydrate 'exposure' according to the discrepancy in phospholipid/glycolipid hydrocarbon chain length. Moreover the finding that this differential effect was not observed in the microtitre assay may indicate that the bilayer width may have some 'pliancy' in liposomes, denied in a surface bilayer film. Thus in liposomes, only the largest chain length discrepancy (e.g. C22:lGb 3 with C14PC) resulted in increased binding. The smaller discrepancies may be accommodated in liposomes by distortion of the bilayer hydrocarbon chain interface. This might also explain the effect of PC chain length on VT binding to renal Gb3. For VT1, only the shortest PC had an enhancing effect on binding in liposomes whereas there was a linear inhibitory effect of increasing chain length on VT binding renal Gb3 in the microtitre assay (Fig. 1A, 1B). Alternatively interdigitation may provide the mechanism of accommodation [46-49]. We have suggested that the differential binding of VT1 and VT2c to different Gb 3 fatty acid homologues is due to a different galabiose conformational requirement for the binding of these two toxins [17]. Using thermodynamic calculations, we identified two possible Gb 3 binding sites per VT B monomer, which accommodated different Gb3 conformers [45]. Many studies have established a contribution of the conformation of carbohydrate hydroxyls in determining the intermolecular organization of bilayer glycosphingolipids [50], and it is therefore not unlikely that the reverse might also be true. Although the bilayer structure of the liposomes can be clearly established by standard electron microscopy (Fig. 2), technical limitations prevent similar analysis of the microtitre supported phospholipid film at present. Phospholipid films however, when hydrated may adopt a bilayer format [46,51] and atomic force electron microscopy [52] would seem ideally suited to confirm this in future studies. The phospholipid film format has often been adopted to quantitate glycolipid receptor binding specificity [53-58]. We have shown that glycolipid receptor function can vary markedly according to the manner of immobilization, particularly when comparing tlc overlay binding with that in a microtitre format with accessory lipids [17, 18]. The biophysical basis for the difference in receptor activity of Gb3 in phospholipid matrices of PCs of different chain length under the conditions we have
described is likely to be complex, requiring consideration of differences in phase separation and possible formation of lipid microdomains, gel/liquid crystalline transition temperatures and molecular area in addition to that of the relative glycolipid/phospholipid acyt chain length. Although such studies are beyond the scope of this report, our present results serve as an initial alert to the potential effect of the solid phase to modify glycolipid receptor function in a phospholipid bilayer. This work also further demonstrates a difference in VT1 and VT2c binding to Gb3. Studies with artificial membranes often assume equivalence of phospholipid bilayers immobilized on a surface and in vesicles [59, 60] and little data counter to this assumption is available. However in a few reports, supported bilayer films have been shown to represent a closer functional in vitro equivalent of natural plasma membranes than liposomal bilayers [61---63]. Our studies provide further evidence that a liposomal bilayer does not necessarily mimic a bilayer on a surface. Our results imply that the lipid composition and local membrane environment in combination, may have a stereoselective effect on glycolipid carbohydrate receptor function. Presentation of glycolipid carbohydrate in a surface bilayer film is not necessarily equivalent to that in a liposome. These studies serve to further illustrate the limitations of a simple mechanism of glycolipid receptor function in a biological membrane [50].
Acknowledgements This work was supported by MRC programme grant no. PG11123. S.A. was supported by a graduate scholarship from the Ministry of Health, Iran. The authors thank Drs J. Boggs, HSC and C. Grant University of Western Ontario, for comments on the manuscript.
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